microRNAs (miRNAs) are a class of small non-coding RNAs known to function in post-transcriptional regulation of gene expression. The goal of this thesis was to characterize the function of known pluripotency-specific miRNAs. Moreover, we extended this study to germ cells and established the miRNA signature of various cell types of mouse spermatogenesis and showed functional relevance for some of the stage-specific miRNAs.
Collectively, we revealed the miRNA profiles of pluripotent cells as well as germ cells and uncovered their function in regulation of pluripotency and spermatogenesis, respectively. The results of this thesis are summarized in the following manuscripts:
3.1. Members of the miR-290 cluster modulate in vitro differentiation of mouse embryonic stem cells.
3.2. Embryonic stem cell-related miRNAs are involved in differentiation of pluripotent cells originating from the germ line.
3.3. MicroRNA signature in various cell types of mouse spermatogenesis: Evidence for stage-specifically expressed miRNA-221, -203, and -34b-5p mediated spermatogenesis regulation.
Each paragraph within the following results section contains a brief description of the aim of the study in context of the complete thesis, the status of each manuscript as well as the author contributions.
Results
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3.1 Members of the miR-290 cluster modulate in vitro differentiation of mouse embryonic stem cells
Recent studies have highlighted the importance of miRNAs, especially miR-290 family members, in maintenance of self-renewal and proliferation properties of pluripotent stem cells. In the first part of this thesis, we tested the possible function of miRNA-290 members during embryonic stem cell (ESC) differentiation. Towards this end, we performed miRNA gain-of-function and loss-of-function experiments in ESCs. The overexpression of miR-290 cluster in ESCs could not prevent the downregulation of Oct4, a pluripotency marker gene, and also failed to sustain the stemness during induced differentiation. However, the suppression of miRNA-290 members expression resulted in an immediate downregulation of Oct4 and cells showed precocious differentiation. Additionally, differentiation towards mesoderm and germ cell lineage was found to be significantly affected in miR-290 cluster overexpressing cells. Reciprocally, the miR-290 cluster suppression resulted in preferential derivation of mesoderm and germ cell lineage cell types. Further studies revealed that miRNA-290 members target Dkk1, a Wnt-signaling inhibitor, to modulate the mesoderm and germ cells differentiation of ESCs. Collectively, our results demonstrate for the first time that pluripotent-specific miRNAs regulate the differentiation of ESCs.
Authors: Athanasios Zovoilis*, Lukasz Smorag*, Angeliki Pantazi, Wolfgang Engel.
* equal contribution to the work
Published in Differentiation. 2009 Sep-Oct;78(2-3):69-78. doi: 10.1016/j.diff.2009.06.003.
Epub 2009 Jul 22.
Results
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3.2. Embryonic stem cell-related miRNAs are involved in differentiation of pluripotent cells originating from the germ line.
We have identified that the expression of miR-290 as well as miR-302 clusters is identical between undifferentiated pluripotent cells originating either from inner cell mass of the blastocyst (ESCs) or from germ cells (multipotent adult germline stem cells (maGSCs)).
However, we noted that maGSCs retain high expression levels of miR-290 cluster, which is associated with high Oct4 expression, during induced differentiation. In the second part of this thesis, we investigated the potential differences between ESCs and maGSCs as well as embryonic germ cells (EGCs) during differentiation and the relevance of miRNAs in this process. We found that undifferentiated maGSCs and EGCs express high levels of germ cell specific marker genes such as Dppa3 and Stra8 compared to ESCs. These expression levels were highly persistent even during the differentiation of maGSCs and EGCs. In light of these findings, we identified that ESCs but not maGSCs retains high expression levels of miRNA-302 cluster during differentiation and inversely correlates with the levels of early-germ cell marker genes. Finally as a proof of concept, we show that overexpression of miR-302 in maGSCs can suppress the preferential differentiation into germ cell lineage during differentiation.
Authors: Athanasios Zovoilis*, Angeliki Pantazi*, Lukasz Smorag*, Lennart Opitz, Gabriela Salinas Riester, Marieke Wolf, Anna Holubowska, Ulrich Zechner, Stewart Colin, Wolfgang Engel
* equal contribution to the work
Published in Mol Hum Reprod. 2010 Nov;16(11):793-803. doi: 10.1093/molehr/gaq053.
Epub 2010 Jun 21.
Results
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3.3. MicroRNA signature in various cell types of mouse spermatogenesis: Evidence for stage-specifically expressed miRNA-221, -203, and -34b-5p mediated spermatogenesis regulation.
Spermatogenesis, the process of haploid male gametes generation, is a complex process regulated by both transcriptional and post-transcriptional mechanisms. To better understand how post-transcriptional mechanisms mediated by miRNAs regulate the self-renewal as well as differentiation process of spermatogonial stem cells (SSCs), we analyzed the miRNA expression profile of various cell types of mouse spermatogenesis. To achieve this goal, we generated a transgenic mouse model (Stra8/EGFP and Sycp3/DsRed) in which pre-meiotic (PrM) and meiotic cells were marked by EGFP and DsRed, respectively. The isolation of pure germ cell populations, i.e., SSC, PrM and meiotic cells and the subsequent miRNA microarray expression analysis led us to establish the miRNA signature of individual cell type. Through functional studies, we show that miRNA-221 regulates the self-renewal of SSCs by targeting c-Kit, while miRNA-203 and -34b-5p function in preventing the precocious activation or repression of germ cell differentiation by targeting Rbm44 and Cdk6, respectively. Taken together, through our data we suggest a working model where spatiotemporal expression of miRNA functions in the regulation of spermatogenesis.
Authors: Lukasz Smorag, Ying Zheng, Jessica Nolte, Ulrich Zechner, Wolfgang Engel, D.V. Krishna Pantakani.
Published in Biol Cell. 2012 Nov;104(11):677-92. doi: 10.1111/boc.201200014. Epub 2012 Sep 24.
Discussion
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